The genome sequence of the common earthworm, Lumbricus terrestris (Linnaeus, 1758)

We present a genome assembly from an individual Lumbricus terrestris (the common earthworm; Annelida; Clitellata; Haplotaxida; Lumbricidae). The genome sequence is 1,056.5 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.93 kilobases in length.


Background
The Darwin Tree of Life project aims to sequence to reference quality the genomes of species of Ireland and Britain (Blaxter et al., 2022).While most species we target make their homes on these islands, some are ecosystem engineers that shape the environments in which they and others live.One such group is the lumbricid annelids, or earthworms, which collectively generate and maintain soils by processing and degrading mineral particles, cycling biological material into the soil, aerating and draining soil through their burrows and providing rich habitats for soil microbes in their casts (Kooch & Jalilvand, 2008).The importance of earthworms as not just inhabitants but prime generators of soil was noted by Charles Darwin in his last book "The Formation of Vegetable Mould Through the Action of Worms, with Observations on their Habits" (Darwin, 1881).
Lumbricus terrestris, the common earthworm (see (NBN Atlas Partnership, 2023) was described by Linnaeus in his "Systema Naturae" in 1758 (genus #246, on pages 647-648 in a paraphyletic "Vermes") (Linnaeus, 1758).Now placed in Lumbricidae within the oligochaete annelids (phylum Annelida, class Oligochaeta, order Crassiclitellata), L. terrestris is the largest of the earthworms of Ireland and Britain, and has, since Darwin, been the focus of intensive study especially in the areas of ecotoxicology and ecotoxicogenomics (Brulle et al., 2010;Sheppard, 1998;Sturzenbaum et al., 2009).As L. terrestris, like other earthworms, carries out respiratory exchange through its skin, and consumes and processes soil matter through its gut, it is particularly exposed to geogenic and anthropogenic contamination of soils (Stürzenbaum et al., 2004).Here we present a chromosomally-complete assembly of L. terrestris, collected from the Wellcome Sanger Institute campus, to support ongoing work on this keystone species.

Genome sequence report
The genome was sequenced from one Lumbricus terrestris (Figure 1) collected from Wellcome Genome Campus, Hinxton, UK (52.08, 0.18).We generated 33-fold coverage using Pacific Biosciences single-molecule HiFi long reads.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 199 missing joins or mis-joins and removed 221 haplotypic duplications, reducing the assembly length by 11.9% and the scaffold number by 33.33%, and decreasing the scaffold N50 by 4.45%.
The final assembly has a total length of 1056.5 Mb in 361 sequence scaffolds with a scaffold N50 of 56.6 Mb (Table 1).
The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (98.77%) of the assembly sequence was assigned to 18 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/6398.

Sample acquisition and nucleic acid extraction
Lumbricus terrestris specimens were collected from Wellcome Genome Campus (latitude 52.08, longitude 0.18) on 2020-03-12.The specimen was taken from soil in woodland by turning leaflitter and subsoil.The collectors were Mark Blaxter (Wellcome Sanger Institute), Peter Kille (University of Cardiff) and David Spurgeon (UK Centre for Ecology & Hydrology) and David Spurgeon identified the specimen.The specimen was flash frozen in liquid nitrogen.One specimen  was used for DNA sequencing and Hi-C data (specimen ID SAN0001206, ToLID wcLumTerr1) and another specimen (specimen ID SAN0001207, ToLID wcLumTerr2) was used for RNA sequencing.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The wcLumTerr1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Bodywall and intestinal tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30.Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample.
The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.
Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from tissue of wcLumTerr2 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina HiSeq 4000 (RNA-Seq) instruments.Hi-C data were also generated from body wall and intestinal tissue of wcLumTerr1 using the Arima2 kit and sequenced on the HiSeq X Ten and Illumina NovaSeq 6000 instrument.et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This   Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Yoshiaki Morino
Institute of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan The paper reports genome sequencing of the common earthworm, Lumbricus terrestris.The chromosome-level assembly was generated using standard methods and is sufficiently high quality.The significance of the species is well introduced.I have only two minor comments: Concerning the absence of sex chromosomes, it would be beneficial to include a mention in the manuscript that earthworms, including this species, are generally hermaphrodites. 1.
The manuscript should describe the source tissues used for the RNA-seq.2.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Marine invertebrate, Evo-Devo I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Lumbricus terrestris, wcLumTerr1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 1,056,547,181 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (98,895,420 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (56,588,617 and 43,887,615 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the metazoa_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/wcLumTerr1.1/dataset/CATKHU01/snail.

Figure 3 .
Figure 3. Genome assembly of Lumbricus terrestris, wcLumTerr1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/wcLumTerr1.1/dataset/CATKHU01/blob.

Figure 5 .
Figure 5. Genome assembly of Lumbricus terrestris, wcLumTerr1.1:Hi-C contact map of the wcLumTerr1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=OfTjARz6QZKy250DRKimLg.

Figure 4 .
Figure 4. Genome assembly of Lumbricus terrestris, wcLumTerr1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/wcLumTerr1.1/dataset/CATKHU01/ cumulative.

Darwin Tree of Life Project Sampling Code of Practice', which
can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
https://doi.org/10.21956/wellcomeopenres.22341.r71560